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Abstract

Dapivirine, a nonnucleoside reverse transcriptase inhibitor, is in development as a microbicide for the protection of women against HIV infection. A randomized, double-blind, phase 1 trial was conducted in 36 healthy HIV-negative women to compare the pharmacokinetics of 2 dapivirine vaginal gel formulations (0.05% each) and their safety with the hydroxyethyl cellulose-based universal placebo gel. Gel was self-administered once daily for a total of 11 days. Blood and vaginal fluid samples were collected sequentially over 24 days for pharmacokinetic analysis. Safety was evaluated by pelvic examination, colposcopy, adverse events, and clinical laboratory assessments. Adverse event profiles were similar for the 3 gels. Most events were mild and not related to study gel. Headache and vaginal hemorrhage (any vaginal bleeding) were most common. Plasma concentrations of dapivirine did not exceed 1.1 ng/mL. Steady-state conditions were reached within approximately 10 days. Dapivirine concentrations in vaginal fluids were slightly higher for Gel 4789, but Cmax values on days 1 and 14 were not significantly different. Terminal half-life was 72-73 hours in plasma and 15-17 hours in vaginal fluids. Both formulations of dapivirine gel were safe and well tolerated. Dapivirine was delivered to the lower genital tract at concentrations at least 5 logs greater than in vitro inhibitory concentrations.

INTRODUCTION

Most individuals infected with HIV (67%) live in sub-Saharan Africa.1 In certain sub-Saharan countries, HIV prevalence among 15- to 24-year-olds is approximately 3 to 4 times greater in women than men.1 As a female-initiated method, vaginal microbicides may be an important option for protecting women from HIV infection. Vaginal microbicide gels can be self-administered and have been shown to have high acceptability rates2-8 and to be easy to use.3-8

Dapivirine (TMC120), a nonnucleoside reverse transcriptase inhibitor (NNRTI), is a microbicide candidate currently in development in multiple vaginal dosage forms including gels and rings.9-15 Dapivirine vaginal gels and rings have been tested in phase 1 and phase 1/2 clinical safety trials.2,4,16,17 Dapivirine is a potent inhibitor of HIV-1 replication in vitro and in vivo9,14,15 and exhibits potent antiviral activity against multiple clades of HIV and both wild-type virus and strains harboring various resistance-inducing mutations.9,15

This article describes the results of a phase 1 study of the pharmacokinetics of 2 dapivirine gel formulations used once daily for 11 days by healthy HIV-negative women. In addition, the safety of Gel 4750 and Gel 4789 was compared with the safety of the hydroxyethyl cellulose (HEC)-based universal placebo gel.18,19

METHODS

Study Design

A phase 1, randomized, double-blind, placebo-controlled trial was conducted in Antwerp, Belgium, in 36 healthy HIV-negative women 18 to 40 years of age to evaluate and compare the pharmacokinetics of 2 dapivirine vaginal gels (Gel 4750 and Gel 4789, both 0.05%) and to assess the safety of these gels compared with the universal placebo gel. All participants provided informed consent at screening (up to 28 days before enrollment on day 1). Study procedures were conducted in accordance with the ethical standards of the responsible committee on human experimentation (Commissie voor Medische Ethiek) and with the Helsinki Declaration of 1975, as revised in 2000.

All participants had negative urine pregnancy test results at enrollment. Women were willing to abstain from sexual activity and the use of vaginal products for the duration of the study. Women used either oral hormonal therapy to avoid menstruation during the study or were on long-acting progestins or progestin-releasing IUDs for at least 6 months before enrollment.

Thirty-six women were randomly assigned (1:1:1) to 1 of 3 vaginal gels (Gel 4750, Gel 4789, or placebo gel), which they used once daily for 11 days (day 1 and days 5 through 14). The date of randomization was planned so that menses did not occur during the 11-day gel administration period. Blood and vaginal fluid samples were collected at each study visit, and cervicovaginal tissue biopsies were collected once from each participant for measurement of dapivirine concentrations as indicated in Figure 1. Safety was assessed by pelvic examinations, clinical laboratory tests (hematology, clinical chemistry, and urinalysis), and adverse events (including clinically significant laboratory abnormalities).

Safety Assessments

Pelvic examinations were performed at screening and on days 1, 2, 5, 15, 16, and 24. Colposcopy was done on days 1, 15, and 24. Blood samples for hematology and clinical chemistry were collected at screening (baseline) and on days 15 and 24. Specimens for urinalysis and cervicovaginal swabs were collected at screening and on days 1 (baseline), 15, and 24. Adverse event data were collected at each study visit and graded according to the National Institutes of Health Division of AIDS (DAIDS) Table for Grading the Severity of Adverse Events.20 Testing for sexually transmitted infections (gonorrhea, chlamydia, and trichomonas) was done at screening and on days 15 and 24, and HIV testing was done at screening and on day 24. Specimen collection, handling, and processing were performed according to the standard operating procedures of the local laboratory.

Blood samples and vaginal fluid samples for measurement of dapivirine concentrations were collected sequentially over a 24-hour period on days 1-2; on days 5, 9, and 11; sequentially over a 32-hour period on days 14-15; and on days 16, 17, 19, 21, and 24 as indicated in Figure 1. Tissue biopsies were collected once on days 2, 15, 16, or 24. The bioanalysis was performed by PRA International, Early Development Services, Zuidlaren, the Netherlands, using high-performance liquid chromatography with tandem-mass spectrometric detection. The analytical methods met the criteria in CFR 320.29, and the assay validation was conducted according to the Food and Drug Administration Guidance for Industry, Bioanalytical Method Validation (May 2001). The pharmacokinetic analysis was performed by Kinesis Pharma B.V., Breda, the Netherlands.

Plasma

Blood samples were collected by venipuncture and stored on ice for up to 1 hour before centrifugation at 1500g for 10 minutes at 4°C. Plasma was aliquotted into polypropylene tubes and frozen at −70°C until shipment on dry ice to PRA International for bioanalysis. At PRA, plasma samples were thawed at room temperature, homogenized, and then centrifuged for 5 minutes at 1500g and 20°C. A 200-μL aliquot was transferred to a glass tube, and 50 μL internal standard solution (2000 pg/mL dapivirine-d4 in water:methanol, 50:50 vol/vol) were added. After vortex mixing, 400 μL of 1 M ammonia buffer (pH 9.2) were added followed by 3 mL of extraction solvent (n-chlorobutane). Samples were mixed vigorously for 2 minutes and then centrifuged for 5 minutes at 2500g and 20°C. The aqueous layer was frozen in an acetone/dry ice bath, and the organic layer was decanted into a 5-mL glass tube. Ten percent 1,2-propanediol solution (100 μL) was added to each tube. The organic layer was evaporated to dryness at 60°C under a nitrogen stream. One hundred microliters of reconstitution solvent (0.1% formic acid:acetonitrile, 70:30 vol/vol) were added to each tube, samples were vortex mixed, and then centrifuged for 1 minute at 1500g and 20°C. Extracts were transferred to autosampler vials. Twenty microliters of sample were injected into the chromatographic system, which consisted of mobile phase A (0.4% ammonia in water) and mobile phase B (acetonitrile) at 40:60 vol/vol with a column temperature of 40°C and a flow rate of 0.300 milliliters per minute. The calibration range for the bioanalytical assay was 3-2000 pg/mL.

Vaginal Fluids

Fluid samples were collected on Alpha Med Tear Test Ophthalmic Strips (AlphaMed Inc., Wauwatosa, WI) at 3 vaginal lumen locations (introitus, mid vagina, and cervix) at the time points indicated in Figure 1. Before sampling, each strip was placed in a tube and weighed on a calibrated analytical balance. Using forceps, the strip was then removed from the tube and placed in the vaginal lumen at the designated location for approximately 2 minutes or until damp up to the indicator mark. The strip was returned to the same tube and reweighed. After weighing, tubes containing samples were immediately frozen at −20°C. Samples were later shipped to PRA International on dry ice for performance of the bioanalytical assay.

At PRA, 2 mL of acetonitrile were added to sample tubes followed by 10 μL of internal standard solution (10.0 μg/mL dapivirine-d4). Samples were vortex mixed, 2 mL of 0.4% ammonia in water were added, and samples were remixed. The extract was transferred to an injection vial, and a 5-μL aliquot was injected into the chromatographic system. Chromatographic conditions consisted of mobile phase A (0.4% ammonia in water) and mobile phase B (acetonitrile) at 40:60 vol/vol% with a column temperature of 40°C and a flow rate of 0.300 milliliters per minute. The calibration range for the assay was 0.400 to 500 nanograms per strip.

Tissues

Participants were randomized using a stratified block design within study groups at a 1:1:1:1 ratio (3 participants per time point) to have biopsies taken on day 2 (24 hours post dose on day 1), day 15 (24 hours post dose on day 14), day 16 (48 hours post dose on day 14), or day 24 (240 hours post dose on day 14). Three full-thickness biopsies (measuring approximately 2 × 4 mm) were collected under local anesthesia (xylocaine spray) from the vagina (introitus and mid vagina) and cervix. If minor bleeding occurred after biopsy, silver nitrate was to be applied locally. Samples were shipped on dry ice to PRA and stored frozen until bioanalysis.

At PRA, samples were to be thawed and weighed in the original tube. Tissue was to be transferred to a clean tube, and the original tube was to be reweighed empty. Methanol (50 μL) was added to each sample followed by 300 μL of each water and acetonitrile. Fifty microliters of internal standard (1.00 μg/mL dapivirine-d4 in acetonitrile) were added to samples, which were then vortex mixed. Tissues were homogenized for 5 minutes in an ice bath and then transferred to a glass extraction tube. One hundred microliters of 25% ammonia were added to each sample followed by 3 mL of chlorobutane. Tubes were tumble mixed for 10 minutes at 30 rpm, then centrifuged for 5 minutes at 3800g and 20°C. The aqueous layer was frozen in an acetone/dry ice bath, and the organic layer was decanted into a 5-mL glass tube and evaporated to dryness at 50°C under a nitrogen stream. Five hundred microliters of reconstitution solvent (0.1% formic acid:acetonitrile, 70:30 vol/vol) were added to each tube, and samples were vortex mixed. Extracts were transferred to autosampler vials, and 2 μL of each sample were injected into the chromatographic system. Chromatographic conditions consisted of mobile phase A (0.4% ammonia in water) and mobile phase B (acetonitrile) at 40:60 vol/vol with a column temperature of 40°C and a flow rate of 0.300 milliliters per minute. The calibration range for the bioanalytical assay was 0.5-250 ng or 10-5000 ng/mL.

Statistics

All observations were included in the statistical analyses. Pharmacokinetic analyses were performed based on actual sampling times. The least square (LS) means of the primary parameters for each treatment group were estimated with a linear mixed effects model, controlling for treatment as fixed effects. A 90% confidence interval (CI) was constructed around the difference between the LS means of test (Gel 4750) and reference (Gel 4789). Both the difference between the LS means and the 90% CI limits were retransformed to the original scale. Treatment effects were considered significant at the 5% level. Statistical equivalence could be shown when the 90% CI limits were within the bioequivalence limits of 80%-125%. As this was a pharmacokinetic study, a total of 36 participants (12 per study group) was considered adequate to allow for meaningful conclusions to be drawn.

RESULTS

Demographic Data

Thirty-six, healthy, HIV-negative women were enrolled in the study. Mean ages were 30.5, 29.5, and 28.9 years, in the Gel 4750, Gel 4789, and placebo gel groups, respectively. All participants were white.

Disposition/Exposure

All randomized participants completed the study. Based on the results of a questionnaire administered on day 14, 2 participants, one in the Gel 4750 group and one in the placebo group, missed a single gel dose (days 9 and 11, respectively).

Safety Data

Adverse Events

No serious adverse events were reported during the study. Most participants (10 to 12 of 12) in each study group had at least 1 treatment-emergent adverse event (TEAE). Headache was reported most often and occurred in 5 to 8 of 12 participants in each study group. No TEAEs were assessed as definitely related to study gel. A greater number of participants in the dapivirine gel groups (8 and 9 of 12) than placebo group (5 of 12) had TEAEs assessed as possibly or probably related to study gel (Table 1). Most possibly/probably related events (12 of 16 MedDRA preferred terms) occurred in only 1 of 12 participants in a single study group. The severity of all but 2 TEAEs was grade 1. Two grade 2 TEAEs were reported in the Gel 4789 group by 2 participants: pyrexia (days 1-5, not related) and headache (days 14-15, possibly related).

Pelvic/Colposcopic Examinations

Two participants had new post-baseline findings during pelvic/colposcopic examination. The findings were reported as TEAEs (grade 1, possibly related). One participant in the Gel 4750 group had cervical erythema (>10 mm) from days 15 to 23, and one participant in the Gel 4789 group had a herpes ulcer on her vulva (<5 mm) from days 14 to 20.

Safety Laboratory Tests

Treatment-emergent hematology, biochemistry, and urinalysis laboratory results outside the reference range of the local laboratory are shown in Table 2. None were considered to be clinically significant according to DAIDS criteria. “Bacteria in urine” was the most common treatment-emergent laboratory abnormality, occurring in 4 to 7 of 12 participants in each study group. The remaining participants in each study group were positive at baseline.

More placebo (7 of 12) than dapivirine (3 of 24) gel users had positive vaginal swab results, with white blood cells being the most common finding. Abnormal swab results were assessed by the investigator as not clinically significant.

No positive test results for trichomonas, gonorrhea, or chlamydia were obtained during the study. There were no HIV seroconversions during the study.

Dapivirine Concentrations

Plasma

During the 24-hour sampling period after administration of a single dose of study gel on day 1, mean dapivirine concentrations in plasma increased more rapidly in the Gel 4750 group, but mean concentrations were comparable at 24 hours (243 vs. 251 pg/mL in the Gel 4750 and 4789 groups, respectively; Fig. 2A). On day 1, median tmax was 23.5 hours in both dapivirine groups (Table 3); however, the maximum concentration may not have been reached at 24 hours because no additional blood draws were scheduled until day 5. Four days after the initial gel dose, mean dapivirine concentrations were 24%-45% of day 1 Cmax values. During the continuous dosing period from days 5 to 14, mean pre-dose concentrations in both dapivirine gel groups increased by ≤10% between days 9 and 11 and between days 11 and 14, suggesting that concentrations were close to steady state.

After gel administration on day 14, mean Cmax and AUC0-24h values in both dapivirine gel groups were 2- to 3-fold greater than day 1 values (Table 3). During the 10-day period after the final gel administration on day 14, mean plasma concentrations declined similarly for the 2 formulations (Fig. 2B). The slopes of semilogarithmic plots of plasma concentration vs. time were similar for both dapivirine gels, indicating that the rate of plasma elimination was comparable. Terminal half-life was longer for Gel 4750 than Gel 4789 (90 vs. 73 hours, Table 3); however, the mean for the Gel 4750 group was skewed by one participant's value. Excluding this participant from the calculation yielded a mean half-life of 72 hours, which was comparable to the value for the Gel 4789 group.

Statistical analysis of pharmacokinetic parameters on days 1 and 14 showed that LS mean ratios for Cmax and AUC0-24h were not significantly different for the 2 gels (P > 0.08). However, equivalence could not be shown because the 90% CIs did not fall entirely within the 80%-125% bioequivalence limits. Interparticipant variability was 29%-30% for both gel groups on day 1, and 15%-22% in the Gel 4750 group vs. 34%-38% in the Gel 4789 group on day 14.

Vaginal Fluids at Introitus, Mid Vagina, and Cervix

Mean dapivirine concentrations in vaginal fluids collected at the introitus, mid vagina, and cervix were in the range of 62-265 μg/g on day 1 (Fig. 3A). Mean concentrations were higher in the Gel 4789 group at each sampling site at both time points on day 1 and remained higher in the Gel 4789 group throughout the study.

In vaginal fluids collected near the cervix on day 1, mean Cmax and AUC0-24h values were more than 2 times greater in the Gel 4789 group than Gel 4750 group (Table 4). Statistical analysis of LS mean ratios showed a significant difference in these pharmacokinetic parameters between the 2 gel groups (P = 0.0247 and 0.0233, respectively). Similar trends were observed in vaginal fluids collected at the introitus and mid vagina, although the differences between study groups did not reach statistical significance.

On day 5, 4 days after initial gel administration, mean dapivirine concentrations in vaginal fluids near the cervix were <10 μg/g in both study groups (Table 4). During the continuous dosing period from days 5 to 14, dapivirine concentrations seemed to reach maximum levels on day 11 and changed <3% from days 11 to 14 (pre-dose). After gel administration on day 14, dapivirine concentrations increased by 58%-62% compared with pre-dose values on day 14. Terminal half-life was 16-17 hours in vaginal fluids.

Similar trends were observed in vaginal fluids collected at the introitus and mid vagina. Statistical analysis of LS mean ratios showed a significant difference between the 2 gel groups in Cmax and AUC0-24h values on day 14 for fluids collected at the mid vagina (P = 0.0442 and 0.0470, respectively) and in day 14 Cmin values for fluids collected near the cervix (P = 0.0242).

Elimination of dapivirine from vaginal fluids collected near the cervix after the last gel administration on day 14 is shown in Figure 3B. During the initial 24-hour period post dose, dapivirine concentrations decreased more rapidly in the Gel 4750 group than in the Gel 4789 group; however, by 48 hours post dose, dapivirine concentrations had decreased to a similar extent in both groups (to 22%-24% of Cmax; Table 4).

On both day 1 and day 14, interparticipant variability in pharmacokinetic parameters was greater in the Gel 4750 group (58%-70% and 41%-91%, respectively) than in the Gel 4789 group (38%-52% and 25%-61%, respectively).

Cervicovaginal Tissues

By error, tissue masses were not reported for biopsied samples collected for assessment of dapivirine concentrations. Therefore, no accurate cervicovaginal tissue concentrations could be calculated. Dapivirine was detected in tissue samples collected at 24 hours post dose on day 1 and at 24 and 48 hours after administration of the last dose on day 14. Dapivirine was not detected in tissue samples collected at 240 hours after the last dose.

Acceptability Questionnaire

All participants responded to an acceptability questionnaire on day 14. More users of Gel 4750 and 4789 (92%, 92%) than universal placebo gel (75%) were willing or very willing to use the gel again in the future. All but one gel user (placebo) found the gel easy or very easy to insert.

Gel leakage after insertion was reported by a greater percentage of placebo users (92%) than dapivirine gel users (Gel 4750, 83%; Gel 4789, 67%). For the majority of participants reporting gel leakage (76%), the leakage was described as slight or requiring the use of a panty liner. Two-thirds of participants in each group responded that the leakage would not prevent use of the gel in the future.

DISCUSSION

Dapivirine vaginal microbicide Gel 4750 and Gel 4789 were safe, well tolerated, and comparable to the HEC-based universal placebo gel in terms of safety. This finding is in agreement with the results of previous safety studies where adverse event rates were similar between dapivirine gel and matching or universal placebo gel.2,4 There were no serious adverse events (SAEs), and headache was the most common TEAE, occurring in 42%-67% of participants in each study group.

One participant showed signs of local inflammation (cervix erythema, Gel 4750 group). Histological and subclinical inflammatory changes were not evaluated in this study. In 2 studies with earlier formulations of dapivirine gel, microscopic changes to the cervix (petechiae, acetowhite finding, erythema) were reported in 6%-17% of participants who used dapivirine gel (0.001%, 0.002%, 0.005%, or 0.02%) or placebo gel (HEC-based universal or matching). In preclinical studies, no evidence of inflammatory effects was seen during 10 days of intravaginal administration of Gel 4789 in rabbits. In addition, intravaginal administration of Gel 4789 and Gel 4759, which differs from Gel 4750 only by the absence of vitamin E TPGS, to mice for 14 days resulted in no changes in innate immunity as measured by inflammatory cells, cytokines, and chemokines in vaginal lavages.

Although the results of several safety laboratory tests were flagged as outside the reference range of the local laboratory, it is important to note that none of these values were considered clinically significant based on DAIDS criteria. When compared with the laboratory test results in 3 earlier studies with lower dapivirine gel doses (0.001%, 0.002%, 0.005%, and 0.02%), no trends were observed between abnormal hematology test results and dapivirine gel dose. Low total bilirubin was noted here and in one earlier study in 8 of 12 participants who used 0.02% dapivirine gel and in 3 of 12 participants who used HEC placebo gel2; however, group means at baseline were at or below the lower limit of the reference ranges in both studies.

The finding of bacteria in urine in 4 to 7 of 12 participants in each study group was considered not clinically significant because sterile procedures were not used for urine collection in this study. Furthermore, 5 to 8 of 12 participants in each study group were positive for bacteria in urine at baseline. Bacteria were not detected in the urine of participants in an earlier study of 0.001%, 0.005%, and 0.02% dapivirine gel (A. M. Nel, MBChB, PhD, in press, 2010).

More users of placebo than dapivirine gel had abnormal vaginal swab results; however, the findings were not clinically significant. Because of the small number of participants in each study group, it is not possible to determine whether dapivirine gel was protective. The frequency of vaginal procedures used in this study could have contributed to the nonclinically significant variability in vaginal swab test results. In a previous placebo-controlled study of dapivirine gel (0.001%, 0.002%, and 0.005%),2 white blood cells were present in vaginal swabs across all study groups (6%-13%), which is similar to what was reported here in the dapivirine groups (8%).

A central premise to the microbicide concept is that elevated levels of potent antiretroviral (ARV) compounds in the genital tract will prevent sexual transmission of HIV. Dapivirine concentrations in vaginal fluids near the cervix in both gel groups (day 14 Cmax 147-231 μg/g) were 4-5 logs greater than the 50% inhibitory concentration (IC50) for dapivirine against HIV-1 in vitro (0.33-2.0 ng/mL)9 and 3 logs greater than plasma concentrations at which at least a 10-fold reduction in viral load was seen in 80%-100% of HIV-positive women after oral administration of 50-100 mg dapivirine twice daily for 7 days (104-208 ng/mL).21 Furthermore, dapivirine levels remained elevated in vaginal fluids for over 24 hours after the last multiple dose, a finding that supports a daily dosing regimen.

An important issue with microbicide use is the potential for development and transmission of virus resistant to the ARV used in the dosage form. Development of resistance in an HIV-positive woman using an ARV-based microbicide could theoretically occur if subtherapeutic drug levels consistent with the selection of resistance in vivo are attained. In this study, the day 14 Cmax values in vaginal fluids near the cervix achieved with both gel formulations (147-231 μg/g) were 6-7 logs greater than the in vitro concentration at which NNRTI resistance mutation Y181C22 was detected using single viral genome analysis (1/32 clones at 0.003 ng/mL; 3/23 clones at 0.03 ng/mL).23 These fluid levels were also 3 to 5 logs higher than the in vitro-derived IC50 values against NNRTI-resistant virus (0.30-18.7 ng/mL).9 Thus, dapivirine concentrations achieved in vaginal fluids with both gels are not likely to be conducive to the transmission or selection of resistant virus. Plasma levels of dapivirine achieved with both gels (day 1 and 14 Cmax values of 0.3 and 0.7 ng/mL, respectively) were 5 logs lower than the levels in vaginal fluids. Importantly, the plasma levels observed with both gels were 3-4 logs lower than what was measured after oral administration of 200-500 mg dapivirine twice daily for 14 days (Cmax 1.5-3.6 μg/mL)24 and therefore are unlikely to inhibit viral replication. The potential role of resistance as a safety concern with microbicide use remains an unresolved issue, and extrapolation from in vitro data to the in vivo situation is not yet possible. Therefore, it is our intention to continue to study this phenomenon in the clinical context through the implementation of seroconvertor trials into which women who become HIV positive during the course of the microbicide trial will be enrolled for further evaluation.

In conclusion, Gel 4750 and Gel 4789 were safe and well tolerated with low systemic absorption. Both gels achieved dapivirine concentrations in vaginal fluids that were over 3 logs greater than plasma levels that resulted in a significant reduction in viral load with oral dapivirine. Further development of either vaginal gel as an HIV microbicide is appropriate.

ACKNOWLEDGMENTS

We wish to thank the principal investigator, Dr. Wouter Haazen, and the co-investigators, Dr. Eva Vets and Dr. Sofie Mesens. We are grateful to the clinical research team at SGS Life Science Services-Clinical Research, to PRA International for bioanalysis, to Kinesis Pharma B.V. for pharmacokinetic analyses and report writing, and to DF/Net Research, Inc. for data management.

Journal of Chromatography B-Analytical Technologies in the Biomedical and Life SciencesDevelopment and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experimentsdas Neves, J; Sarmento, B; Amiji, M; Bahia, MFJournal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 911():
76-83.10.1016/j.jchromb.2012.10.034CrossRef

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